Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis
- Dalhousie University, Canada
- Gonzaga University, United States
Figures
Figure 1
Structure and function of ubiquinone and rhodoquinone in mitochondria.
(A) Under aerobic conditions, electrons from NADH or succinate (Suc) are shuttled to ubiquinone (UQ) via Complex I (I) or Complex II (II) respectively to generate NAD (NAD+), fumarate (fum) and ubiquinol (UQH2). The electron transfer via Complex I fuels the transport of protons from the mitochondrial matrix into the intermembrane space (IMS). Electrons from UQH2 are transferred to Complex III, cytochrome c, and ultimately O2 via Complex IV with the concomitant pumping of protons. Complex V (V) uses this proton gradient to synthesize ATP. (B) In anaerobic eukaryotes, electrons from NADH are shuttled to rhodoquinone (RQ) to generate rhodoquinol (RH2). The RQ pool is regenerated via CII functioning as a fumarate reductase.
Figure 2 with 3 supplements
Maximum-likelihood phylogeny of RquA proteins constructed from an alignment of homologs from 166 organisms and 197 aligned sites.
Eukaryotic proteins are coloured based on their phylogenetic affiliations: Obazoa (purple), Stramenopiles-Alveolata (orange), Excavata (green), Amoebozoa (magenta) and Rhizaria (blue). Hexagons represent taxa where RQ has been detected experimentally. Proteobacterial designations (α,β,γ) are indicated in the grey squares. Genetic linkage of rquA and genes related to respiratory function (complex I-IV, cytochrome c metabolism, or heme metabolism) are shown with chain-links and detailed in Supplementary file 1. When these indications are in a collapsed node, the number of genomes showing linkage are shown in brackets. Bootstrap values (or posterior probability) greater than 70 (0.7) and 90 (0.9) are shown with open circles (squares) or closed circles (squares) respectively. The presence of spliceosomal introns in the eukaryotic sequences are indicated with ‘i' in a box. Dashed branches were made shorter by 50% to facilitate visualization.
Figure 2—figure supplement 1
Intron positions in eukaryotic rquA gene sequences.
Nucleotide from both transcriptome and genomic sequencing projects (when available) were aligned using Sequencher (Gene Codes v 5.4.6) and introns were manually inspected for position, length and phase. The phase indicates the position of the intron relative to the codon where phase 0, 1 and 2 begin before the codon, in between position 1 and 2, or in between position 2 and 3 respectively. All introns were major spliceosomal introns with GT-AG boundaries. In general, intron positions and lengths were not conserved in distantly related species.
Figure 2—figure supplement 2
Phyletic distribution of RQUA among alphaproteobacteria superimposed on the alphaproteobacterial species tree.
Maximum likelihood analysis of a 200 phylum-specific phylogenetic marker genes representing 54,400 sites from 210 representative alphaproteobacteria under the LG + C60 + F (PMSF)+ Γ4 model of evolution implemented in IQ-TREE. Tree was rooted using an outgroup of other proteobacteria. Nodes with maximal support are unlabeled, while those with support values between 95–99 are labeled with squares. RQUA-containing taxa are coloured in purple or orange representing Group A and Group B type RQUA respectively. Orders of alphaproteobacteria are indicated on the right of the tree with a summary of the number of alphaproteobacterial genomes interrogated for RQUA presence/absence in Genbank (green box).
Figure 2—figure supplement 3
RquA homologues lack critical S-adenosyl methionine (SAM) binding site.
Conserved residues known to interact with the carbonyl group of SAM are shown with black triangles. A substitution from aspartate to glutamine was obversed in all bacterial (represented here by Rhodospirillum rubrum) and eukaryotic RquA demonstrated with a open circle. Taxa are coloured with super-Group Affiliation as in Figure 2. Motif features: [VILFG]-[LIVCS]-[DENLV]-[VALMIT]-[GLYCFA]-[CSTAYFPAG]-[GA]-[PTSGNKRMV]-[GD]).
Figure 3
RquA co-occurs with quinone-utilizing enzymes.
Eukaryotic genomes and transcriptomes were surveyed for homologs of respiratory chain components (Complexes I-V, CI-CV), alternative oxidase (AOX), dihydroorotate dehydrogenase (DHOH), electron-transferring flavoprotein system (ETF), glycerol-3-phosphate dehydrogenase (G3P), sulfite:quinone oxidoreductase (SQO), RquA, and one or more anaerobiosis-associated protein (AAP; detailed in Supplementary file 1). Grey and white circles indicate that homologs were not detected in transcriptome and genome sequence data respectively. Half circle in CI for Pygsuia biforma and Trichomonas vaginalis indicates only two subunits (NUOE and NUOF) were identified. ‘ψ ‘indicates pseudogenes.
Figure 4 with 3 supplements
Subcellular localization of RquA and rhodoquinone production in Pygsuia biforma.
(A) Antibodies raised against RquA (green) colocalized with MitoTracker (red). Confocal slices (0.3 μm) were deconvoluted (using a constrained interative algorithm) and combined to render a 3D image. DAPI stained nuclei (blue) were volume rendered in Imaris for clarity. (B) Lipid extracts were separated by liquid chromatography and analyzed with selected-reaction monitoring mass spectrometry. Rhodoquinone species eluted from the column in roughly 3 min intervals as chain length increases (RQ8-10). Diagnostic product ions corresponding to the rhodoquinone head group (182.1 m/z) following fragmentation of parent ions were detected.
Figure 4—figure supplement 1
Antibodies directed against Pygsuia RquA (PbRquA) recognize purified recombinant PbRquA by western blot analysis.
Proteins isolated from whole cell extracts of E. coli induced to express pGEX4T-1 (1) and pGEX- Pb-rquA (2) and glutathione-S-transferase(GST)-Pb-rquA purified with glutathione magnetic beads (3) were resolved by SDS-PAGE and probed by immunoblotting using anti- PbRquA. The estimated molecular weight of the GST-PbRquA is 56 kDa (i.e., 26 kDa GST and 30 kDa for PbRquA). The antibody interacted with an endogenous E. coli protein (1 and 2, lower signal) and this signal was mostly reduced upon purification of the protein (3).
Figure 4—figure supplement 2
Spectra represent the elution times of detection of the rhodoquinone head group (182.1 m/z; left) and ubiquinone head group (197.1 m/z) following fragmentation of parent ions of different isoprenyl chain lengths as indicated.
https://doi.org/10.7554/eLife.34292.014
Figure 4—figure supplement 3
Lipid extracts were separated by high-performance liquid chromatography and analyzed with mixed-reaction monitoring mass spectrometry from Pygsuia mixed culture, Pygsuia ‘food’ bacteria culture, glassware, and Rhodospirillum rubrum as indicated.
Spectra represent the elution times of detection of the rhodoquinone head group (182.1 m/z; left) and ubiquinone head group (197.1 m/z) following fragmentation of parent ions of different isoprenyl chain lengths as indicated.
Figure 5
The interactions of rhodoquinone with other mitochondrial redox reactions in different eukaryotes with mitochondrion-related organelles.
Standard reduction potentials for each major reaction involved in rhodoquinone (R)) and ubiquinone (U) metabolism are shown in increasing order of potential. Half reaction equations are detailed in supplementary file 1. The electron transfer is more favourable when passed to a species with a more positive standard reduction potential (i.e. from left to right). Abbreviations: I, Complex I; II, Complex II; III, Complex III; IV, Complex IV; V, Complex V; E, electron transferring flavoprotein dehydrogenase; G, glycerol-3-phosphate (G3P) dehydrogenase; A alternative oxidase; Fum, fumarate; Suc, succinate; DHAP, dihydroxyacetone phosphate; and Cyt c, cytrochrome c. U* Indicates that the involvement of ubiquinone is unknown, ? indicates the direction of electron transfer is unknown. Absence of a circle indicates that no homologs were detected in the organism. Genes undergoing pseudogenization are shown in white cirlces.
Author response image 1
Cumulative frequency plot of transcriptome completeness for the marine microbial eukaryotes sequencing projects.
https://doi.org/10.7554/eLife.34292.023Tables
Table 1
Approximate unbiased topology tests for RquA analyses.
https://doi.org/10.7554/eLife.34292.007| Monophyly Tested | Tree file | CONSEL p-AUa |
|---|---|---|
| Group A and Group B | ||
| Maximum likelihood tree | Figure 2; Supplementary file 2 - tree 2 | 0.743 |
| Group A eukaryotes + Group B eukaryotes | Supplementary file 2 - tree 3 | 3.00E-37*** |
| Group A1 eukaryotes: Blastocystis, Proteromonas, Neoparamoebids, Euglenids, Pygsuia | Supplementary file 2 - tree 4 | 0.622 |
| Group A1 eukaryotes + Brevimastigamonas | Supplementary file 2 - tree 5 | 0.46 |
| Group A1 eukaryotes + Brevimastigamonas + Mastigamoeba | Supplementary file 2 - tree 6 | 0.294 |
| Group A eukaryotes | Supplementary file 2 - tree 7 | 0.253 |
| Group B eukaryotes | Supplementary file 2 - tree 8 | 0.179 |
| Obazoa (Pygsuia + Monosiga) | Supplementary file 2 - tree 9 | 0.002** |
| Amoerphea (Obazoa + Amoebozoa) | Supplementary file 2 - tree 10 | 1.00E-32*** |
| Amoebozoa | Supplementary file 2 - tree 11 | 0.206 |
| Stramenopiles + Alveolates | Supplementary file 2 - tree 12 | 0.004** |
| Stramenopiles + Alveolates + Rhizaria (SAR) | Supplementary file 2 - tree 13 | 0.034* |
| Diaphoretickes (SAR + Euglenids) | Supplementary file 2 - tree 14 | 0.018* |
| Rhizaria | Supplementary file 2 - tree 15 | 1.00E-60*** |
| Eukaryotes + MAG alphaproteobacteria | Supplementary file 2 - tree 16 | 2.00E-41*** |
| Group A eukaryotes + MAG alphaproteobacteria | Supplementary file 2 - tree 17 | 0.227 |
| Alphaproteobacteria | Supplementary file 2 - tree 18 | 5.00E-34*** |
| Eukaryotes + alphaproteobacteria | Supplementary file 2 - tree 19 | 8.00E-43*** |
| Group A eukaryotes + Group A alphaproteobacteria | Supplementary file 2 - tree 20 | 3.00E-31*** |
| Group B eukaryotes + Group B alphaproteobacteria | Supplementary file 2 - tree 21 | 4.00E-05*** |
| Group A | ||
| Maximum likelihood tree | Supplementary file 3 - tree 1 | 0.892 |
| Eukaryotes | Supplementary file 3 - tree 2 | 0.225 |
| Amoebozoa | Supplementary file 3 - tree 3 | 0.315 |
| Pygsuia + Amoebozoa (Amorphea) | Supplementary file 3 - tree 4 | 0.22 |
| Eukaryotes + alphaproteobacteria | Supplementary file 3 - tree 5 | 3.00E-59*** |
| Eukaryotes + MAG alphaproteobacteria | Supplementary file 3 - tree 6 | 0.226 |
| Group B | ||
| Maximum likelihood tree | Supplementary file 4 - tree 1 | 0.827 |
| Eukaryotes | Supplementary file 4 - tree 2 | 0.081 |
| Stramenopiles + Alveolates | Supplementary file 4 - tree 3 | 0.287 |
| SAR | Supplementary file 4 - tree 4 | 0.281 |
| Eukaryotes + alphaproteobacteria | Supplementary file 4 - tree 5 | 2.00E-75*** |
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aitalicized values indicate topologies that could not be rejected (p<0.05).
* 0.05 > p > 0.01
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** 0.01 > p > 0.001
*** p < 0.001
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Table 1—source data 1
Topology test output from CONSEL.
Trees 1-20 represent trees 2-21 from Supplementary file 2; trees 21-120 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html
- https://doi.org/10.7554/eLife.34292.008
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Table 1—source data 2
Topology test output from CONSEL.
Trees 1-6 represent trees1-6 from Supplementary file 3; trees 7-106 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html
- https://doi.org/10.7554/eLife.34292.009
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Table 1—source data 3
Topology test output from CONSEL.
Trees 1-5 represent trees 1-5 from Supplementary file 4; trees 6-105 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html
- https://doi.org/10.7554/eLife.34292.010
Author response table 1
Numbers of complete genomes and transcriptome projects surveyed from each of the major lineage of eukaryotes.
https://doi.org/10.7554/eLife.34292.024RquA detecteda | NCBI Genomesb | NCIB TSA nrc | MMETSPd | |
| Alveolata | 7 | 105 | 32 | 134 |
| Amoebozoa | 4 | 41 | 10 | 8 |
| Apusozoa | 0 | 1 | 0 | 0 |
| Breviatea | 1 | 1 | 1 | 0 |
| Centroheliozoa | 0 | 0 | 1 | 0 |
| Cryptophyta | 0 | 8 | 1 | 25 |
| Euglenozoa | 2e | 55 | 3 | 5 |
| Fornicata | 0 | 2 | 2 | 0 |
| Glaucocystophyceae | 0 | 4 | 0 | 3 |
| Haptophyceae | 0 | 5 | 2 | 61 |
| Heterolobosea | 0 | 5 | 1 | 2 |
| Jakobida | 0 | 6 | 0 | 0 |
| Malawimonadidae | 0 | 2 | 0 | 0 |
| Opisthokonta | 1 | 9,240 | 1395 | 10 |
| Oxymonadida | 0 | 1 | 1 | 0 |
| Parabasalia | 0 | 2 | 1 | 0 |
| Rhizaria | 4 | 12 | 4 | 21 |
| Rhodophyta | 0 | 124 | 15 | 8 |
| Stramenopiles | 5 | 150 | 49 | 279 |
| Viridiplantae | 0 | 2,461 | 499 | 72 |
| unclassified | 0 | 0 | 3 | 19 |
| Multispecies with undefined taxa ID | 0 | 0 | 82 | 19 |
| Total | 24 | 12225 | 2102 | 647 |
| a Source: this study b Source: NCBI Taxonomy Browser, selecting “Genomes” – January 28, 2018 c Source NCBI Trace Archive for the transcriptome shotgun assembly database availble at https://www.ncbi.nlm.nih.gov/Traces/wgs/?term=tsa Taxonomy ID were extracted and taxonomy parsed with ete-toolkit. Only non-redundant taxonomy IDs are shown. d Source: iMicrobe; Taxonomy ID were extracted and taxonomy assigned with ete-toolkit. Only non-redundant taxonomy IDs are shown.e Three copies were identified in Eutriptiella and 1 in Euglena | ||||
Additional files
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Supplementary file 1
Excel file with workbooks containing information about the number of genomes surveyed, PFAM domains of putative RquA homologues, gene accession numbers for RquA and Q-utilizing proteins, mitochondrial targeting sequence information, redox half-potentials used for Figure 5, bacterial genes with and without linkage to respiratory complexes.
- https://doi.org/10.7554/eLife.34292.017
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Supplementary file 2
PDF with all the trees for the full RquA phylogenetic analysis and associated topology tests
- https://doi.org/10.7554/eLife.34292.018
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Supplementary file 3
PDF with all the tree for the Group A phylogenetic analysis and associated topology tests
- https://doi.org/10.7554/eLife.34292.019
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Supplementary file 4
PDF with all the tree for the Group B phylogenetic analysis and associated topology tests
- https://doi.org/10.7554/eLife.34292.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.34292.021
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Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis
eLife 7:e34292.
https://doi.org/10.7554/eLife.34292
